Membrane lipid renovation in Pseudomonas aeruginosa ‐ implications for phage therapy?

Abstract Pseudomonas aeruginosa is an important Gram‐negative pathogen with intrinsic resistance to many clinically used antibiotics. It is particularly troublesome in nosocomial infections, immunocompromised patients, and individuals with cystic fibrosis. Antimicrobial resistance (AMR) is a huge threat to global health, with a predicted 10 million people dying from resistant infections by 2050. A promising therapy for combatting AMR infections is phage therapy. However, more research is required to investigate mechanisms that may influence the efficacy of phage therapy. An important overlooked aspect is the impact of membrane lipid remodelling on phage binding ability. P. aeruginosa undergoes changes in membrane lipids when it encounters phosphorus stress, an environmental perturbation that is likely to occur during infection. Lipid changes include the substitution of glycerophospholipids with surrogate glycolipids and the over‐production of ornithine‐containing aminolipids. Given that membrane lipids are known to influence the structure and function of membrane proteins, we propose that changes in the composition of membrane lipids during infection may alter phage binding and subsequent phage infection dynamics. Consideration of such effects needs to be urgently prioritised in order to develop the most effective phage therapy strategies for P. aeruginosa infections.


Abstract
Pseudomonas aeruginosa is an important Gram-negative pathogen with intrinsic resistance to many clinically used antibiotics. It is particularly troublesome in nosocomial infections, immunocompromised patients, and individuals with cystic fibrosis. Antimicrobial resistance (AMR) is a huge threat to global health, with a predicted 10 million people dying from resistant infections by 2050. A promising therapy for combatting AMR infections is phage therapy. However, more research is required to investigate mechanisms that may influence the efficacy of phage therapy. An important overlooked aspect is the impact of membrane lipid remodelling on phage binding ability. P. aeruginosa undergoes changes in membrane lipids when it encounters phosphorus stress, an environmental perturbation that is likely to occur during infection. Lipid changes include the substitution of glycerophospholipids with surrogate glycolipids and the over-production of ornithine-containing aminolipids. Given that membrane lipids are known to influence the structure and function of membrane proteins, we propose that changes in the composition of membrane lipids during infection may alter phage binding and subsequent phage infection dynamics. Consideration of such effects needs to be urgently prioritised in order to develop the most effective phage therapy strategies for P. aeruginosa infections.

THE IMPORTANCE OF PSEUDOMONAS AERUGINOSA AS A PATHOGEN
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. It is a major pathogen in hospitals, being found on various surfaces and in water supplies with the potential to infect immunocompromised and vulnerable patients (Kizny Gordon et al., 2017;Gellatly and Hancock, 2013). Common infection sites for P. aeruginosa include burns and wounds, the urinary tract, bloodstream and the lungs (Morin et al., 2021). P. aeruginosa is especially associated with lung infections in those patients with cystic fibrosis (CF) and chronic obstructive pulmonary disease (Welp and Bomberger, 2020). In Europe, around 41% of adults with CF have chronic P. aeruginosa infection (Orenti et al., 2022) which is associated with increased morbidity and mortality (Jurado-Martín et al., 2021).
CF is a genetic disease caused by mutations in a chloride ion channel present in the membranes of cells of the lungs, gut and pancreas (Scoffone et al., 2017). This causes abnormally thick mucus, which is difficult to clear from the lungs. As a result, pathogens that get into the lung are not cleared, leading to infection.
Respiratory disease is the main cause of death in people with cystic fibrosis (Martin et al., 2016). Lung infections in CF patients caused by P. aeruginosa begin as recurrent, intermittent infections, but, as time goes on, they progress to become chronic infections (Folkesson et al., 2012;Maldonado et al., 2016). There is evidence that an undetected reservoir of P. aeruginosa exists in the nasal sinuses of supposedly recovered patients, and it is from this reservoir that their lungs become reinfected (Hansen et al., 2012). The chronic inflammatory response to persistent P. aeruginosa infection leads to serious damage to lung tissue (Folkesson et al., 2012). As a result, individuals with CF have a much lower life expectancy than the general population, with babies born in the UK in 2020 expected to live to a median age of 47 (Keogh et al., 2019). This is comparable to a European study which showed that the median survival age for patients in the European Cystic Fibrosis Patients cohort is 51.7 (McKone et al., 2021).
P. aeruginosa infections of CF lungs persist despite high levels of antimicrobial therapy given to infected individuals. One of the contributing factors for this is that P. aeruginosa possesses several intrinsic antimicrobial resistance (AMR) mechanisms (Jurado-Martín et al., 2021;Moradali et al., 2017), along with other adaptations to the CF lung environment detailed later in this review. P. aeruginosa possesses a Gram-negative outer membrane that is highly impermeable, restricting the entry of antibiotics into the cell. If antibiotics do penetrate this barrier, it possesses genome-encoded efflux pumps that can expel antibiotics (Shigemura et al., 2015;Dreier and Ruggerone, 2015). Further mechanisms of AMR can be acquired by horizontal gene transfer from other bacterial species or spontaneous mutation (Breidenstein et al., 2011) with examples including β-lactamases (Llanes et al., 2013;Pang et al., 2019) or quinolone resistance genes (Araujo et al., 2016;Cavalcanti et al., 2015). During infection P. aeruginosa also forms biofilms, which are aggregates of cells within an extracellular matrix of exopolysaccharides, extracellular DNA, and proteins. These structures increase both resistance to antibiotics and to the host immune system (Pang et al., 2019;Billings et al., 2013;Taylor et al., 2014).
Due to the multitude of AMR strategies employed by P. aeruginosa, treatment with antimicrobial therapy has become significantly less effective across the world (Al-Orphaly et al., 2021). As such, the World Health Organisation (WHO) has now recognised that carbapenem-resistant P. aeruginosa is a global threat to human health (WHO, 2017), highlighting the importance of identifying alternative treatment strategies for P. aeruginosa infections, such as phage therapy. The focus of this review is to highlight the challenges posed to phage therapy in light of the recent discovery of lipid renovation in the physiological adaptation of P. aeruginosa to phosphorus limitation during infection (Jones et al., 2021). Thus, we briefly discuss current understanding of the P. aeruginosa lipid membrane as well as the status of P. aeruginosa phage research, including the isolation of novel phages and characterisation of their receptors. However, readers are also encouraged to consult excellent recent reviews on AMR mechanisms (Pang et al., 2019), cystic fibrosis (Rossi et al., 2021;Malhotra et al., 2019) and membrane lipids (Kondokova et al., 2015;Sohlenkamp and Geiger, 2016), which will only be briefly touched upon where relevant in this review.

P. AERUGINOSA MEMBRANES AND THEIR LIPIDS
The P. aeruginosa outer membrane, like in other Gramnegative bacteria, is asymmetric (Figure 1), with the inner leaflet comprising largely glycerophospholipids (GP) and the outer leaflet containing a high concentration of lipopolysaccharide (LPS). LPS has three components: the lipid A anchor, which sits within the membrane, the core oligosaccharide, which is attached to lipid A, and the O oligosaccharide, or O antigen, which is attached to the core oligosaccharide (Needham and Trent, 2013).
The lipid A moiety is made up of hydrophobic acyl chains linked to a backbone glucosamine dimer by ester or amide bonds. The number of acyl chains can vary depending on the species and environmental conditions, but in P. aeruginosa there are typically four acyl chains (Maldonado et al., 2016). Lipid A is recognised by Tolllike receptor 4 (TLR4)-MD2, which triggers an inflammation response in order to try and clear the bacteria (Ciesielska et al., 2021). Some P. aeruginosa strains have no O antigen in their LPS, and this is known as "rough-type" LPS (whereas LPS with O antigen is "smooth") (Maldonado et al., 2016). All components of the LPS can undergo modification under different conditions. For example the removal, addition, or modification of phosphate groups on LPS can alter the charge of the membrane, influencing susceptibility to cationic antimicrobial peptides (CAMPs) (Powers and Trent, 2018).
Besides LPS, the main lipids in membranes of Pseudomonas species are GP, of which the most common are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and phosphatidylcholine (PC) ( Table 1) (Kondakova et al., 2015). PE and PG make up the vast majority of GP in the membrane, but during stationary phase growth CL can accumulate to up to 10% of all GP (Kondakova et al., 2015). Different lipid species have different charges and functions within the membrane (Sohlenkamp and Geiger, 2016). PE, the most abundant GP in Pseudomonas membranes is zwitterionic at pH 7 (Sohlenkamp and Geiger, 2016), and is important in maintaining membrane structure by increasing lateral pressure and introducing curvature stress (Kondakova et al., 2015). PE is also a precursor to a number of essential biological molecules such as diacyl glycerol (DAG), fatty acids, phosphatidic acid (PA) and LPS (Gibellini and Smith, 2010). At pH 7 PC is also zwitterionic, whereas PG and CL are both anionic. PG can form intermolecular H bonds within the membrane (Zhao et al., 2008), which is important for membrane stability. CL is synthesised by the condensation of two PG molecules and plays a role in the formation of dynamic protein-lipid membrane domains with high curvature, such as at sites of bacterial division (Mileykovskaya and Dowhan, 2005).
Modifications to membrane lipids can occur in response to environmental conditions (Geiger et al., 2010;Klein et al., 2009). An example of this is the addition of amino acids (Figure 1), such as the addition of lysine to PG to form lysyl-PG, which has been observed in P. aeruginosa, and can increase resistance to CAMPs (Geiger et al., 2010). PG can also be modified with alanine in P. aeruginosa under acidic growth conditions, which can increase resistance to certain antimicrobials e.g. Cr 3+ , and the osmolyte sodium lactate (Klein et al., 2009). (1) In low phosphate conditions the enzyme Phospholipase C (PlcP) removes the phosphate group from phospholipids to leave diacylglycerol (DAG), from which non-phosphoruscontaining lipids such as MGDG and GADG can be formed (Jones et al., 2021).
(2) In normal growth conditions ornithine lipid (OL) accounts for 2-15% of total lipids, but during low phosphate conditions, or interaction with lung epithelium, the OL level increases .
(3) P. aeruginosa has been shown to modify its membrane lipids through addition of amino acids. PG can be modified with alanine or lysine, which can increase resistance to antimicrobials (Geiger et al., 2010;Klein et al., 2009). (4) P. aeruginosa cells from chronic lung infections in people with CF have little or no O antigen on their LPS (Maldonado et al., 2016). (5) LPS is also modified by addition of the positively charged aminoarabinose and phosphoethanolamine to the lipid A part of the molecule (Edrington et al., 2011). (6) Acyl chains are also added to lipid A such as the fatty acid palmitate, and secondary acyl chains can be added to the fatty acids. The 3-position fatty acid may also be removed (Maldonado et al., 2016). (7) Cardiolipin (CL) stabilises aquaporin Z, a tetrameric water efflux channel, and also increases the transport of water through the channel (Laganowsky et al., 2014). (8) Outer membrane porin F (OmpF) contains LPS binding sites and forms complexes with LPS molecules. Mutation of one of these sites prevented LPS binding and stopped OmpF forming a trimer (Arunmanee et al., 2016).
Under low phosphate conditions P. aeruginosa upregulates the olsBA operon which synthesises the phosphate-free ornithine lipid (OL) (Lewenza et al., 2011;Jones et al., 2021). We have recently shown that under phosphorus-limited conditions P. aeruginosa substitutes membrane phospholipids with the non-phosphorus containing glyceroglycolipids monoglucosyldiacylglycerol (MGDG) and glucuronic acid diacylglycerol (GADG) ( Table 1). The synthesis of glyceroglycolipids in P. aeruginosa is carried out by a two-step pathway, involving a metallophospholipase PlcP and two glycosyltransferases, denoted Agt1 and Agt2 (Jones et al., 2021). P. aeruginosa has been shown to upregulate the olsBA operon, plcP, agt1, and agt2 upon interaction with human epithelial cells (Frisk et al., 2004;Chugani and Greenberg, 2007), and in sputum samples from CF patients P. aeruginosa upregulates olsBA and agt2 (Rossi et al., 2018). Low phosphate levels can also occur in the serum postoperatively or following major burns and is associated T A B L E 1 Characteristics of major membrane lipids found in P. aeruginosa

Lipid
Structure Charge at pH 7

Glucuronic acid diacylglycerol (GADG) Anionic
Ornithine lipid (OL) Zwitterionic with worse clinical outcomes (Sadot et al., 2019;Loghmani et al., 2010). Hypophosphatemia is especially prevalent in intensive care units (Chen et al., 2021). Therefore, studying the response of P. aeruginosa to low phosphate levels is important to understanding its infection biology.

ADAPTATION OF P. AERUGINOSA TO THE CF LUNG ENVIRONMENT
Most new P. aeruginosa infections that occur in individuals with CF come from the environment, rather than from the lungs of other infected individuals (Yang et al., 2011). On entry to the lung, P. aeruginosa needs to adapt to complex and variable micro-environments in order to survive (Folkesson et al., 2012;Garg et al., 2017). Pressures encountered include osmotic stress due to the presence of high viscosity mucus, varying oxygen levels, and reactive oxygen and nitrogen species produced by the inflammatory response (Winstanley et al., 2016;Sommer et al., 2020;Bhagirath et al., 2016). CF patients will also likely be being treated long term with a variety of antibiotics (Winstanley et al., 2016), which by definition presents a challenge to P. aeruginosa. Bacterial adaptation involves several key strategies, including modification of lipid A, changes in membrane lipid composition and the formation of biofilms (Needham and Trent, 2013;Frisk et al., 2004;Yang et al., 2011). Soon after colonisation of the CF airway, lipid A of P. aeruginosa is modified through both the addition of the positively charged amino sugar residue aminoarabinose and the addition of palmitate ( Figure 1) (Ernst et al., 2007). Aminoarabinose alters the charge of lipid A, increasing resistance to innate immune elements including CAMPs and complement. The addition of the fatty acid palmitate (Figure 1) is catalysed by the PagP enzyme which is under the control of the PhoPQ regulatory system (Thaipisuttikul et al., 2014). The addition of palmitate increases membrane integrity and decreases TLR4 activation (Needham and Trent, 2013). Another modification under the control of PhoPQ is the removal of the 3-position fatty acid by PagL, which has been shown to occur in P. aeruginosa (Ernst et al., 2006). Under-acylation of lipid A is known to lower the inflammatory response (Di Lorenzo et al., 2015). P. aeruginosa can also add secondary acyl chains to the fatty acids of lipid A (Maldonado et al., 2016).
P. aeruginosa cells in the early stage of infection have complete O antigens, but those from chronic lung infections in CF patients have a rough LPS phenotype, with short or no O-antigens ( Figure 1) (Maldonado et al., 2016). Loss of the O-antigen seems to confer an advantage as it is immunogenic, and therefore cells lacking it will be less likely to be detected and destroyed by the immune system. However, cells lacking the O-antigen are also less virulent (Kintz and Goldberg, 2008).
Adapting to the CF lung environment also involves changes to membrane lipids (Naughton et al., 2011;Frisk et al., 2004). The cardiolipin synthase gene has been shown to be upregulated in P. aeruginosa during chronic infection of the CF lung (Naughton et al., 2011), indicating increased proportions of CL in the membrane. Addition of amino acids to phospholipids may also occur during infection (see section 2), and can increase resistance to CAMPs, bacteriocins, and antibiotics.
Another lipid which is altered in infection is OL. In low phosphate environments (e.g Gao et al., 2004), or on interaction with the lung epithelium (Frisk et al., 2004;Chugani and Greenberg, 2007), the percentage of OLs in the membrane massively increases (Figure 1). With increased levels of OLs the charge and hydrophobicity of the membrane changes and the susceptibility of P. aeruginosa to CAMPs and antibiotics is reduced. Binding of macrophages is lessened, and biofilm formation is enhanced . It has been shown that OLs decrease the expression of two macrophage enzymes involved in inflammation . Together, this suggests that increased OL contributes to the persistence of P. aeruginosa in chronic infection.
Interestingly, our own recent data suggests that membrane lipid renovation in P. aeruginosa in response to phosphorus stress during lung infection also confers elevated resistance to antimicrobial peptides. Indeed, these surrogate glyceroglycolipids increase resistance to polymyxin B in P. aeruginosa as well as recombinant Escherichia coli strains overexpressing glyceroglycolipids (Jones et al., 2021). This newly discovered lipid renovation strategy could potentially play an important role in the adaptation of P. aeruginosa during lung infection. Analysis of metatranscriptomic datasets from sputum samples taken from CF patients showed overproduction of PlcP and Agt, two of the key enzymes responsible for the formation of the glyceroglycolipids MGDG and GADG (Figure 1) (Jones et al., 2021), as well as alkaline phosphatase PhoA, suggesting P. aeruginosa is experiencing phosphorus limitation during lung infection.
In the CF lung, biofilm formation can also provide protection for P. aeruginosa against phagocytosis, antibiotics, antibodies, osmotic stress and oxidative stress (Yang et al., 2011). Accumulation of mutations which lead to mucoidy and biofilm formation are seen in P. aeruginosa in CF patients (Ernst et al., 2006), alongside the upregulation of genes important for biofilm formation (Rossi et al., 2020). When biofilms initially form they have a higher diversity of fatty acids in their phospholipids than planktonic cells, and the amount of branched fatty acid chains is increased. However, as the biofilm ages, the diversity decreases again, and they become more like planktonic bacteria again (Benamara et al., 2014). This may be because the biofilm cells are preparing to become planktonic again to seek out a new place to form a biofilm.
The chronic use of antibiotics in patients with CF may also impact the membrane lipids of P. aeruginosa. Cationic peptides can cause clustering of anionic lipids in the membrane, with CL segregating into domains in the presence of CAMPS (Epand et al., 2016). Polymyxin B, an antibiotic of last resort against P. aeruginosa, has been shown to cause lipid exchange between the inner and outer membrane of Gram-negative bacteria (Berglund et al., 2015;Clausell et al., 2007;Yu et al., 2015). Polymyxins (Polymyxin B and Colistin) work by binding to LPS and disrupting the outer membrane, therefore resistance to them usually involves modification of LPS (Mohapatra et al., 2021). The lipid A moieties of polymyxin-resistant P. aeruginosa are modified with aminoarabinose, and the membrane lipid profiles are significantly different from the wild type (Han et al., 2018).

PHAGES INFECTING PSEUDOMONAS AERUGINOSA AND PHAGE THERAPY
Bacteriophages (or phages) are viruses that infect bacteria, and can be found in any natural environment where bacteria are found (Dion et al., 2020). Phages infecting Pseudomonas are widely obtained with 780 phage isolated and sequenced to date (May, 2022) (Cook et al., 2021). They span the diversity of known phage types from ssDNA phages (Invovidiae) and RNA phages (Leviviridae) to the more common dsDNA phages (Myoviridae, Podoviridae, Siphoviridae, Autographviridae, Ackermannviridae). Trials in animals have shown positive outcomes from using phage therapy against P. aeruginosa infections. Mouse (Waters et al., 2017;Morello et al., 2011) and zebrafish (Cafora et al., 2019) models of cystic fibrosis have shown significantly improved survival rates and reduced bacterial load, as have mouse models of other forms of P. aeruginosa infection (McVay et al., 2007;Watanabe et al., 2007). A strength of phage therapy is its ability to target and significantly clear P. aeruginosa biofilm biomass (Fong et al., 2017;Waters et al., 2017), where antibiotics largely fail. In rats, phages have been shown to have a synergistic relationship with the antibiotic ciprofloxacin, resulting in 10,000 times more bacterial clearance than either treatment alone (Oechslin et al., 2017). Human case reports have demonstrated clearance of P. aeruginosa using phage therapy in urinary tract infections, lung infections, infection of an aortic graft, bacteraemia, and more (see Table 2). Wright et al., 2009 demonstrated the safety and efficacy of phage therapy for ear infections caused by P. aeruginosa in a randomised, double-blind, placebocontrolled phase I/II clinical trial (Wright et al., 2009). In another such trial, Jault et al., 2019 investigated a phage cocktail to treat burn wounds infected with P. aeruginosa. However, due to a drop in phage titre after manufacturing, patients were given the cocktail at 4-5 orders of magnitude lower than intended, which did not show efficacy (Jault et al., 2019). Recent trials and case studies of the use of phage therapy to treat P. aeruginosa infections in humans are summarised in Table 2.
In order to infect a bacterium, a phage first binds to its receptor on the bacterial cell surface. The receptor is often a type of lipid-anchored polysaccharide or a membrane protein (Chaturongakul and Ounjai, 2014). Some phages are extremely specific to a single species or strain of bacteria, while others can have a broader host range, and hence the receptor plays an important role in dictating the host range of the phage (de Jonge et al., 2019). While some mechanisms of phage binding are well-characterised e.g. the binding of phage T4 to E. coli (Brzozowska et al., 2018;Washizaki et al., 2016), there is much that is still not known. One particular aspect is how phage binding is affected by the altered membrane lipid composition of its host, if/when this occurs during the infection process, which may alter the orientation, structure or function of a phage receptor. We consider this next with specific regard to P. aeruginosa infections and the development of effective phage therapy strategies.
There are at least 50 P. aeruginosa phages which have had their receptors characterised (Figure 2; Supplementary Table 1). Most of the P. aeruginosa phage receptors are either the type IV pilus or LPS, but at least one phage uses outer membrane porin M (OprM) as its receptor (Chan et al., 2016). There are undoubtedly far more membrane proteins and structures that are used as phage receptors on the surface of P. aeruginosa, as only a small proportion of known P. aeruginosa phages have a receptor identified.

THE IMPACT OF THE LIPID ENVIRONMENT ON PHAGE RECEPTORS AND PHAGE THERAPY
As phage receptors are either membrane proteins, polysaccharides or other membrane structures (Silva et al., 2016), their presence will most likely be influenced by their lipid surroundings. The changes to lipids that occur in P. aeruginosa during adaptation to the CF lung environment, described earlier, including LPS modifications and the production of non-phosphate lipid classes, are very likely to have an impact on membrane properties. As detailed below, the lipid environment of membrane proteins has an important influence on their structure and function. Notably, one major mechanism

2019
Case report Lung AB-PA01 and Navy phage cocktails Yes, piperacillin-tazobactam, tobramycin and colistin 2 lung transplant recipients with multidrug-resistant P. aeruginosa. Clinical improvement seen in both patients compared to antibiotics alone. P. aeruginosa did return in both patients, but in patient two did not return for two months. In both cases isolates showed increased susceptibility to several antibiotic classes. Aslam et al., 2019 (Continues) of phage resistance is through changes to phage receptors, such as masking, removing or modifying them (Olszak et al., 2017). This highlights how even small changes in the receptor can prevent phage binding, and therefore phage infection.
There are certainly precedents that changes in membrane lipids can affect their interaction with membrane proteins (for a summary, see Table 3). For example, the membrane protein aquaporin Z (AqpZ), a tetrameric water efflux channel in E. coli, was shown to be stabilised by membrane lipids, especially by CL (Figure 1). Further experiments showed that CL modulates the function of ApqZ (Laganowsky et al., 2014). The lipid phosphatidylinositol was found to bind to and stabilise the mechanosensitive channel of large conductance (MscL) greater than other lipids, although all lipids tested stabilised MscL to some extent (Laganowsky et al., 2014). AmtB, a trimeric E. coli ammonia channel, is stabilised by CL and PG. Stabilisation of the protein increases linearly with the amount of these lipids added. Further investigation showed that AmtB has selectivity for PG-like head groups. AmtB contains a loop which forms a lipidbinding site with PG or CL, through hydrogen bonds, a water bridge and hydrophobic interactions (Laganowsky et al., 2014). The translocon is a complex of proteins which translocate proteins across the inner membrane of Gram-negative bacteria. It includes transmembrane proteins SecYEG, and cytoplasmic protein SecA, which binds to SecYEG in the process of translocation (Ryabichko et al., 2020). SecYEG requires CL for its stability and efficient function as a high affinity binder of SecA. The interaction of the positively charged N-terminus of SecA with negatively charged membrane lipids primes it allosterically for binding to SecYEG (Ryabichko et al., 2020). PG binds to outer membrane porin F (OmpF), which is made up of three pore channels, and stabilises it in an open that infect Escherichia. It is therefore possible that changes to the levels of PG in the outer membrane could cause conformational changes in OmpF, altering phage adsorption. OmpF has also been shown to contain a number of LPS binding sites, forming complexes with a variable number of LPS molecules (Figure 1). Mutation of one of these binding sites prevented LPS binding and stopped OmpF forming a trimer in vivo (Arunmanee et al., 2016). Interactions between the LPS layer and membrane proteins are likely to be important in the impermeability of the outer membrane (Arunmanee et al., 2016).
The potassium channel KcsA selects for anionic lipids in its core, and these lipids are important for the potassium-conducting function of the protein (Contreras et al., 2011). The interactions between negatively charged phospholipids and positively charged amino acids may help to guide the orientation of membrane proteins (Contreras et al., 2011). An example of this is the interaction of lactose permease (LacY) with the anionic lipids PG and CL in E. coli. The N-terminal helical bundle of LacY can be completely inverted, have a mixed topology, or a fully native topology as the percentage of the zwitterionic PE in the membrane compared to the anionic PG and CL is increased from 0% to 70%. This change in conformation happens due to changes in lipid ratios both at the time of LacY insertion into the membrane, and after insertion (Vitrac et al., 2013).
The presence of aminoacyl phospholipids may also affect the rigidity, fluidity and permeability of the membrane. The presence of aminoacyl phospholipids in vesicles has been shown to stabilise the bilayer and alter peptide binding behaviour (Slavetinsky et al., 2017). Ordinarily, magnesium cations bridge adjacent LPS molecules. Under magnesium-deficient growth conditions, outer membrane protein H (OprH) is upregulated and becomes a major part of the P. aeruginosa outer membrane (Edrington et al., 2011). OprH contains multiple LPS interaction sites allowing it to interact with multiple LPS molecules at once and facilitating the formation of cross-links. In turn, these cross-linkages between LPS molecules increase its stability and decrease membrane permeability (Edrington et al., 2011). OprH is genetically linked to the PhoPQ regulatory system, where the twocomponent system is upregulated in response to magnesium deficiency. LPS alterations, such as the aminoarabinose and palmitate additions, are also regulated by the PhoPQ system. OprH may have a higher affinity for LPS when these modifications are present (Edrington et al., 2011).
These examples demonstrate the importance of membrane lipids in the structure and function of membrane proteins and LPS, both of which can be receptors for P. aeruginosa phages. Therefore, when P. aeruginosa undergoes lipid renovation in response to phosphorus limitation during infection, this will likely have an impact on membrane properties. Comparing the proteomes of wild-type P. aeruginosa and a plcP-  Table 1 deletion mutant, both under phosphorus limitation, revealed several membrane proteins to be differentially expressed depending on the ability to remodel lipids (Jones et al., 2021). This included PilC, a membrane porin (OpdP), and an outer membrane receptor (FptA) (Jones et al., 2021). From these data it is thus conceivable that changes in membrane lipids may have subsequent knock-on effects for phage therapy. This could be due to an impact on general membrane properties following lipid remodelling, or due to direct interactions with a particular protein. As a proof-of-concept, we have observed that phage adsorption efficiency can indeed be affected by lipid remodelling in response to phosphorus limitation (R. Lyon unpublished data).
In conclusion, we argue that it is important to better understand the relationship between the environment, the bacterial cell surface and the subsequent impact on phage receptors and phage absorption. This further links to how changes in membrane lipid composition in response to phosphorus limitation during lung infections may affect the efficacy of phage therapy. Certainly, problems have been encountered translating the success of phage therapy in the laboratory to success in clinical trials (Valente et al., 2021). While this is likely to be the result of many confounding factors, one of these could be the influence of the changing lipid makeup of the bacterial membrane and its subsequent impact on phage receptors. We propose that considering both the native lipid environment and the lipid remodelled membrane while developing a phage cocktail will be important in increasing the likelihood of success of phage therapy when treating P. aeruginosa lung infections.